JUN and EGR-1 are critical components in the apoptosis pathway induced by bortezomib. (A) Western blot demonstrated down-regulation of JUN expression in U266 and OCI-My5 myeloma cell lines transfected with JUN shRNA (JUN-KD) and EGR-1 down-regulation in U266, OCI-My5, and OPM2 cell lines transfected with EGR-1 shRNA (EGR-1-KD) compared with scrambled control shRNA (SCR). (B) JUN binding activity was evaluated in nuclear extracts of U266 and OCI-My5 JUN-KD cell lines by the use of an ELISA-based assay (expressed as relative value compared with each control). Each sample is expressed as the mean ± SEM of 2 duplicates. U266 (C) and OCI-My5 (D) cell lines, with and without shRNA-mediated JUN knockdown (JUN) or EGR-1 knockdown (EGR1), were cultured for 48 hours with 10nM and 20nM bortezomib. Cell viability was determined every 12 hours by trypan blue exclusion. Patients with newly diagnosed MM on TT2 (E) and TT3 (F) were divided into 4 groups based on the expression of JUN with EGR-1. Kaplan-Meier estimates on TT2 show 6-year actuarial probabilities of 43% death in cases with JUN^LEGR1^L, 46% death in cases with JUN^HEGR1^L, 35% death in cases with JUN^LEGR1^H, and 23% death with JUN^HEGR1^H. Kaplan-Meier estimates on TT3 show 3-year actuarial probabilities of 42% death in cases with JUN^LEGR1^L, 4% death in cases with JUN^HEGR1^L, 13% death in cases with JUN^LEGR1^H, and 6% death with JUN^HEGR1^H.